Novel proteins and novel genes encoding the same

ABSTRACT

Novel proteins, novel genes encoding the same, plasmids respectively comprising these genes, transformants respectively comprising these plasmids, antibodies or fragments thereof against the above novel proteins, methods of detecting a bacterial infection, and novel polynucleotides are disclosed. The novel proteins are activated human macrophage-specific proteins.

TECHNICAL FIELD

[0001] The present invention relates to plural novel proteins, a novelgene encoding each of the proteins, a plasmid comprising each of thegenes, a transformant comprising each of the plasmids, an antibody or afragment thereof against each of the novel proteins, a method fordetecting a bacterial infection, and novel polynucleotides. The novelproteins of the present invention are activated humanmacrophage-specific proteins.

BACKGROUND ART

[0002] It is known that a Lipopolysaccharide (LPS) is a glycolipidexisting in an outer membrane of Gram-negative bacterium and activates amacrophage to induce an expression of many genes. Known examples of suchgenes having expressions that are induced by the LPS are those ofinterleukin (IL)-1, exhibiting an antitumor function or an inflammatoryfunction, such as a function to cause an inflammation by a bacterialinfection, IL-6, IL-12, IL-15, IL-18, a tumor necrosis factor (TNF), ora chemokine (such as IL-8 or MCP); granulocyte colony-stimulating factor(G-CSF) exhibiting a hemapoietic function, monocyte (M)-CSF, or GM-CSF;or collagenase playing a main role in an inflammation such as thatcaused by a bacterial infection, cyclooxygenase (COX), or a nitrogenoxide synthase (iNOS) or the like. Almost all of the above proteinsencoded by the above genes are physiologically active proteins playingimportant roles in a body [Annu. Rev. lmmunol., 2, 283-318 (1984);Inflammation: Basic Principles and Clinical Correlates, 637-662, RavenPress Ltd., New York (1992)].

[0003] There are about 0.1 million genes in the human chromosome, butonly 10 or 20 percentage thereof have been isolated and identified.Therefore, as almost all of the genes have not been isolated oranalyzed, it is believed that almost all of the genes having anexpression that is specifically induced by LPS are unidentified novelgenes.

[0004] Septicemia is a systemic disease wherein a festering lesionexists in a body, and many bacteria are intermittently or continuouslyintroduced into the blood from the festering lesion. A diagnosis of thesepticemia is carried out by culturing the blood, and when the existenceof bacteria is proved, the illness is definitely diagnosed as thesepticemia. However, the above method has disadvantages, namely, themethod is time-consuming, and when a blood sample is drawn, it may becontaminated with bacteria from skin, such as Staphylococcusepidermidis.

[0005] Therefore, the inventors of the present invention made anintensive search for genes having an expression that is inducedspecifically at a macrophage by an LPS-stimulation, for the purpose ofan application for developing a new method of diagnosis and/ormedicament for treating a disease such as inflammation, allergy, orcancer, particularly a bacterial infection. As a result, three novelgenes were isolated and identified. Further, the present inventors foundthat these three genes were not expressed in healthy persons, but wereexpressed in patients suffering from a bacterial infection. Theinvention is based on the above findings.

DISCLOSURE OF INVENTION

[0006] The present invention relates to

[0007] (1) a protein comprising an amino acid sequence of SEQ ID NO: 2in the sequence listing, or a variation functionally equivalent thereto,or a fragment of the protein or the variation (hereinafter sometimescollectively referred to as a “first novel protein of the presentinvention”),

[0008] (2) a protein comprising an amino acid sequence of SEQ ID NO: 4in the sequence listing, or a variation functionally equivalent thereto,or a fragment of the protein or the variation (hereinafter sometimescollectively referred to as a “second novel protein of the presentinvention”), and

[0009] (3) a protein comprising an amino acid sequence of SEQ ID NO: 6in the sequence listing, or a variation functionally equivalent thereto,or a fragment of the protein or the variation (hereinafter sometimescollectively referred to as a “third novel protein of the presentinvention”).

[0010] Further, the present invention relates to

[0011] (1) a gene encoding the above-mentioned “first novel protein ofthe present invention” (hereinafter sometimes referred to as a “firstnovel gene of the present invention”),

[0012] (2) a gene encoding the above-mentioned “second novel protein ofthe present invention” (hereinafter sometimes referred to as a “secondnovel gene of the present invention”), and

[0013] (3) a gene encoding the above-mentioned “third novel protein ofthe present invention” (hereinafter sometimes referred to as a “thirdnovel gene of the present invention”).

[0014] Further, the present invention relates to plasmids comprisingeach of the above-mentioned genes.

[0015] Further, the present invention relates to transformantscomprising each of the above-mentioned plasmids.

[0016] Further, the present invention relates to antibodies or fragmentsthereof, characterized by being reactive specifically to each of theabove-mentioned proteins or variations functionally equivalent thereto.

[0017] Further, the present invention relates to a method for detectinga bacterial infection, characterized by analyzing the proteins or thevariations functionally equivalent thereto, or the mRNAs thereof, in asample to be detected.

[0018] Further, the present invention relates to

[0019] (1) a polynucleotide capable of specifically hybridizing to anmRNA consisting of an base sequence of SEQ ID NO: 1 in the sequencelisting (hereinafter sometimes referred to as a “first probe of thepresent invention”),

[0020] (2) a polynucleotide capable of specifically hybridizing to anmRNA consisting of an base sequence of SEQ ID NO: 3 in the sequencelisting (hereinafter sometimes referred to as a “second probe of thepresent invention”), and

[0021] (3) a polynucleotide capable of specifically hybridizing to anmRNA consisting of an base sequence of SEQ ID NO: 5 in the sequencelisting (hereinafter sometimes referred to as a “third probe of thepresent invention”).

[0022] The term “variation functionally equivalent” as used herein meansa protein having an amino acid sequence wherein one or more(particularly one or several) amino acids are deleted in, changed in, oradded to the amino acid sequence of an original protein, and exhibitingthe same activities as the original protein. The term “added” as usedherein includes an addition of one or more (particularly one or several)amino acids to an N-terminus and/or a C-terminus of an amino acidsequence, and an insertion of one or more (particularly one or several)amino acids to an inside of an amino acid sequence.

[0023] Further, the term “homologous protein” as used herein means aprotein comprising an amino acid sequence having a 90% or more(preferably 95% or more, more preferably 98% or more, most preferably99% or more) homology with the amino acid sequence of an originalprotein, and exhibiting the same activities as the original protein. Theterm “homology” as used herein means a value calculated by BLAST [Basiclocal alignment search tool; Altschul, S. F. et al., J. Mol. Biol., 215,403-410, (1990)].

[0024] Furthermore, the terms “gene” and “polynucleotide” as used hereininclude both of DNA and RNA.

BRIEF DESCRIPTION OF DRAWINGS

[0025]FIG. 1 shows the results of electrophoresis wherein the expressionof three novel genes of the present invention in human macrophagesstimulated by LPS or not stimulated by LPS was detected by a northernblotting method.

[0026]FIG. 2 shows the results of electrophoresis wherein thetissue-specific expression of three novel genes of the present inventionwas detected by a northern blotting method.

[0027]FIG. 3 provides micrographs showing the results of the expressionof the gene NLG-1-1 of the present invention in COS-1 cells.

[0028]FIG. 4 provides micrographs showing the results of the expressionof the gene NLG-2 of the present invention in COS-1 cells.

[0029]FIG. 5 provides micrographs showing the results of a FISH analysisof the gene NLG-1-1 of the present invention.

[0030]FIG. 6 provides photographs showing the results of electrophoresiswherein the expression of three novel genes of the present invention inhealthy persons and patients suffering from septicemia was detected by anorthern blotting method.

BEST MODE FOR CARRYING OUT THE INVENTION

[0031] The present invention will be explained in detail hereinafter.

[0032] The first novel protein of the present invention includes

[0033] (1) a protein comprising an amino acid sequence of SEQ ID NO: 2in the sequence listing,

[0034] (2) a variation functionally equivalent to the protein (1),

[0035] (3) a protein homologous to the protein (1), and

[0036] (4) a fragment thereof [i.e., a fragment of the protein (1), thevariation (2), or the homologous protein (3)]. A protein consisting ofthe amino acid sequence of SEQ ID NO: 2 in the sequence listing, or avariation functionally equivalent or protein homologous thereto arepreferred.

[0037] The protein consisting of the amino acid sequence of SEQ ID NO: 2in the sequence listing consists of 481 amino acid residues. The proteinconsisting of the amino acid sequence of SEQ ID NO: 2 in the sequencelisting has a high homology of approximately 82% in the amino acidsequence with mouse IRG-1 (lmmune-responsive protein-1) [Immnogenetics,41, 263-270, (1995)], and thus seems to be human IRG-1.

[0038] In the amino acid sequence of SEQ ID NO: 2 in the sequencelisting, there exist eight known sites to be phospholylated by proteinkinase C and ten known sites to be phospholylated by casein kinase C. Asa result, it is believed that the protein consisting of the amino acidsequence of SEQ ID NO: 2 in the sequence listing plays an important rolein an intracellular signal transduction system which transducesinformation of LPS-stimulation.

[0039] Further, a signal peptide sequence at the N-terminus does notexist, and thus, it is believed that the protein consisting of the aminoacid sequence of SEQ ID NO: 2 in the sequence listing exhibitsbiological activities in cells.

[0040] As the protein comprising the amino acid sequence of SEQ ID NO: 2in the sequence listing, there may be mentioned, for example, a fusionprotein of the protein consisting of the amino acid sequence of SEQ IDNO: 2 in the sequence listing with a fusion partner. In the fusionprotein, the fusion partner may be linked with the N-terminus and/or Cterminus of the protein consisting of the amino acid sequence of SEQ IDNO: 2.

[0041] As the fusion partner, for example, a protein for purificationsuch as the whole or a part of glutathione-S-transferase (GST), aprotein for detection such as the whole or a part of β-galactosidase αpeptide (LacZ α), or a protein for expression such as a signal sequencemay be used.

[0042] Further, in the fusion protein, an amino acid sequence which maybe restrictively digested with a proteolytic enzyme such as thrombin orfactor Xa may be optionally inserted between the protein consisting ofthe amino acid sequence of SEQ ID NO: 2 in the sequence listing and thefusion partner.

[0043] The fragment of the protein comprising the amino acid sequence ofSEQ ID NO: 2 in the sequence listing or the variation functionallyequivalent or protein homologous thereto is not particularly limited, solong as it may be used as an immunogen to prepare the first antibody orfragment thereof according to the present invention, but preferablyconsists of 13 or more amino acid residues, more preferably 20 or moreamino acid residues, most preferably 50 or more amino acid residues.

[0044] The first novel protein of the present invention may be obtainedby various known methods. For example, the protein may be prepared byusing a known genetic engineering technique and the first novel gene ofthe present invention.

[0045] The first novel gene of the present invention is not particularlylimited, so long as it encodes the first novel protein of the presentinvention. As the gene, there may be mentioned, for example, a geneconsisting of the 37th to 1479th bases in the base sequence of SEQ IDNO: 1 in the sequence listing.

[0046] The gene consisting of the 37th to 1479th bases in the basesequence of SEQ ID NO: 1 in the sequence listing encodes the proteinconsisting of the amino acid sequence of SEQ ID NO: 2 in the sequencelisting. Further, the gene consisting of the 37th to 1479th bases in thebase sequence of SEQ ID NO: 1 in the sequence listing is not expressedin healthy persons, but is expressed in patients suffering from abacterial infection.

[0047] The first probe of the present invention is not particularlylimited, so long as it is capable of specifically hybridizing to an mRNAconsisting of the base sequence of SEQ ID NO: 1 in the sequence listing.As the probe, there may be mentioned, for example, a single or doublestranded polynucleotide consisting of a base sequence complementary tothat of SEQ ID NO: 1 in the sequence listing, or a partial base sequencethereof. The lower limit of the number of bases in the first probe ofthe present invention is not particularly limited, but is preferably 18or more, more particularly 26 or more, most particularly 41 or more.Further, the upper limit thereof is not particularly limited, but ispreferably 2180 or less. The expression “specifically hybridize with anmRNA consisting of the base sequence of SEQ ID NO: 1 in the sequencelisting” as used herein means that a polynucleotide does not hybridizewith mRNAs derived from a healthy person, but will hybridize with themRNA consisting of the base sequence of SEQ ID NO: 1 in the sequencelisting, under the conditions described in Example 1(4). In thoseconditions, it is twice washed with 2×SSC (standard sodium citrate)containing 0.1% sodium dodecyl sulfate (SDS) at room temperature for 20minutes, and further twice washed with 0.2×SSC containing 0.1% SDS at65° C. for 20 minutes.

[0048] The second novel protein of the present invention includes

[0049] (1) a protein comprising an amino acid sequence of SEQ ID NO: 4in the sequence listing,

[0050] (2) a variation functionally equivalent to the protein (1),

[0051] (3) a protein homologous to the protein (1), and

[0052] (4) a fragment thereof [i.e., a fragment of the protein (1), thevariation (2), or the homologous protein (3)]. A protein consisting ofthe amino acid sequence of SEQ ID NO: 4 in the sequence listing, or avariation functionally equivalent or protein homologous thereto arepreferred.

[0053] The protein consisting of the amino acid sequence of SEQ ID NO: 4in the sequence listing consists of 390 amino acid residues. The proteinconsisting of the amino acid sequence of SEQ ID NO: 4 in the sequencelisting has a high homology of approximately 82% in the amino acidsequence with mouse IRG-1 (lmmune-responsive protein-1) [Immnogenetics,41, 263-270, (1995)], and thus seems to be human IRG-1.

[0054] In the amino acid sequence of SEQ ID NO: 4 in the sequencelisting, there exist six known sites to be phospholylated by proteinkinase C and eight known sites to be phospholylated by casein kinase C.As a result, it is believed that the protein consisting of the aminoacid sequence of SEQ ID NO: 4 in the sequence listing plays an importantrole in an intracellular signal transduction system which transducesinformation of LPS-stimulation.

[0055] Further, a signal peptide sequence at the N-terminus does notexist, and thus, it is believed that the protein consisting of the aminoacid sequence of SEQ ID NO: 4 in the sequence listing exhibitsbiological activities in cells.

[0056] As the protein comprising the amino acid sequence of SEQ ID NO: 4in the sequence listing, there may be mentioned, for example, a fusionprotein of the protein consisting of the amino acid sequence of SEQ IDNO: 4 in the sequence listing with a fusion partner. In the fusionprotein, the fusion partner may be linked with the N-terminus and/or Cterminus of the protein consisting of the amino acid sequence of SEQ IDNO: 4.

[0057] As the fusion partner, for example, a protein for purificationsuch as the whole or a part of glutathione-S-transferase (GST), aprotein for detection such as the whole or a part of β-galactosidase αpeptide (LacZ α), or a protein for expression such as a signal sequencemay be used.

[0058] Further, in the fusion protein, an amino acid sequence which maybe restrictively digested with a proteolytic enzyme such as thrombin orfactor Xa may be optionally inserted between the protein consisting ofthe amino acid sequence of SEQ ID NO: 4 in the sequence listing and thefusion partner.

[0059] The fragment of the protein comprising the amino acid sequence ofSEQ ID NO: 4 in the sequence listing or the variation functionallyequivalent or protein homologous thereto is not particularly limited, solong as it may be used as an immunogen to prepare the second antibody orfragment thereof according to the present invention, but preferablyconsists of 13 or more amino acid residues, more preferably 20 or moreamino acid residues, most preferably 50 or more amino acid residues.

[0060] The second novel protein of the present invention may be obtainedby various known methods. For example, the protein may be prepared byusing a known genetic engineering technique and the second novel gene ofthe present invention.

[0061] The second novel gene of the present invention is notparticularly limited, so long as it encodes the second novel protein ofthe present invention. As the gene, there may be mentioned, for example,a gene consisting of the 126th to 1295th bases in the base sequence ofSEQ ID NO: 3 in the sequence listing.

[0062] The gene consisting of the 126th to 1295th bases in the basesequence of SEQ ID NO: 3 in the sequence listing encodes the proteinconsisting of the amino acid sequence of SEQ ID NO: 4 in the sequencelisting. Further, the gene consisting of the 126th to 1295th bases inthe base sequence of SEQ ID NO: 3 in the sequence listing is notexpressed in healthy persons, but is expressed in patients sufferingfrom a bacterial infection.

[0063] The second probe of the present invention is not particularlylimited, so long as it is capable of specifically hybridizing to an mRNAconsisting of the base sequence of SEQ ID NO: 3 in the sequence listing.As the probe, there may be mentioned, for example, a single or doublestranded polynucleotide consisting of a base sequence complementary tothat of SEQ ID NO: 3 in the sequence listing, or a partial base sequencethereof. The lower limit of the number of bases in the second probe ofthe present invention is not particularly limited, but is preferably 18or more, more particularly 26 or more, most particularly 41 or more.Further, the upper limit thereof is not particularly limited, but ispreferably 1970 or less. The expression “specifically hybridize with anmRNA consisting of the base sequence of SEQ ID NO: 3 in the sequencelisting” as used herein means that a polynucleotide does not hybridizewith mRNAs derived from a healthy person, but will hybridize with themRNA consisting of the base sequence of SEQ ID NO: 3 in the sequencelisting, under the conditions described in Example 1(4).

[0064] The third novel protein of the present invention includes

[0065] a protein comprising an amino acid sequence of SEQ ID NO: 6 inthe sequence listing,

[0066] (2) a variation functionally equivalent to the protein (1),

[0067] (3) a protein homologous to the protein (1), and

[0068] (4) a fragment thereof [i.e., a fragment of the protein (1), thevariation (2), or the homologous protein (3)]. A protein consisting ofthe amino acid sequence of SEQ ID NO: 6 in the sequence listing, or avariation functionally equivalent or protein homologous thereto arepreferred.

[0069] The protein consisting of the amino acid sequence of SEQ ID NO: 6in the sequence listing consists of 83 amino acid residues. The proteinconsisting of the amino acid sequence of SEQ ID NO: 6 in the sequencelisting has a homology of approximately 27% in the amino acid sequencewith mouse NADH-ubiquinoneoxidoreductase MLRQ subunit (CI-MLRQ). It isreported that the mouse NADH-ubiquinoneoxidoreductase MLRQ subunitexists in the complex I, one of four complexes I, II, III, and IV whichform an electron transport system of a mitochondrion, and takes part ina production of an active oxygen [Circulation Res., 85, 357-363 (1999);Biochem. Mol. Biol. Int., 43, 669-675 (1997)]. The protein consisting ofthe amino acid sequence of SEQ ID NO: 6 in the sequence listing does nothave a gap, as suggested from the amino acid sequence thereof, and it isassumed that the protein has a structure similar to that of the mouseNADH-ubiquinoneoxidoreductase MLRQ subunit. Therefore, the protein maycontain an electron transport activity and take part in a production ofan active oxygen upon inflammation.

[0070] Further, a signal peptide sequence at the N-terminus does notexist, and thus, it is believed that the protein consisting of the aminoacid sequence of SEQ ID NO: 6 in the sequence listing exhibitsbiological activities in cells.

[0071] As the protein comprising the amino acid sequence of SEQ ID NO: 6in the sequence listing, there may be mentioned, for example, a fusionprotein of the protein consisting of the amino acid sequence of SEQ IDNO: 6 in the sequence listing with a fusion partner. In the fusionprotein, the fusion partner may be linked with the N-terminus and/or Cterminus of the protein consisting of the amino acid sequence of SEQ IDNO: 6.

[0072] As the fusion partner, for example, a protein for purificationsuch as the whole or a part of glutathione-S-transferase (GST), aprotein for detection such as the whole or a part of β-galactosidase αpeptide (LacZ α), or a protein for expression such as a signal sequencemay be used.

[0073] Further, in the fusion protein, an amino acid sequence which maybe restrictively digested with a proteolytic enzyme such as thrombin orfactor Xa may be optionally inserted between the protein consisting ofthe amino acid sequence of SEQ ID NO: 6 in the sequence listing and thefusion partner.

[0074] The fragment of the protein comprising the amino acid sequence ofSEQ ID NO: 6 in the sequence listing or the variation functionallyequivalent or protein homologous thereto is not particularly limited, solong as it may be used as an immunogen to prepare the third antibody orfragment thereof according to the present invention, but preferablyconsists of 13 or more amino acid residues, more preferably 20 or moreamino acid residues, most preferably 50 or more amino acid residues.

[0075] The third novel protein of the present invention may be obtainedby various known methods. For example, the protein may be prepared byusing a known genetic engineering technique and the third novel gene ofthe present invention.

[0076] The third novel gene of the present invention is not particularlylimited, so long as it encodes the third novel protein of the presentinvention. As the gene, there may be mentioned, for example, a geneconsisting of the 56th to 304th bases in the base sequence of SEQ ID NO:5 in the sequence listing.

[0077] The gene consisting of the 56th to 1304th bases in the basesequence of SEQ ID NO: 5 in the sequence listing encodes the proteinconsisting of the amino acid sequence of SEQ ID NO: 6 in the sequencelisting. Further, the gene consisting of the 56th to 1304th bases in thebase sequence of SEQ ID NO: 5 in the sequence listing is not expressedin healthy persons, but is expressed in patients suffering from abacterial infection.

[0078] The third probe of the present invention is not particularlylimited, so long as it is capable of specifically hybridizing to an mRNAconsisting of the base sequence of SEQ ID NO: 5 in the sequence listing.As the probe, there may be mentioned, for example, a single or doublestranded polynucleotide consisting of a base sequence complementary tothat of SEQ ID NO: 5 in the sequence listing, or a partial base sequencethereof. The lower limit of the number of bases in the third probe ofthe present invention is not particularly limited, but is preferably 18or more, more particularly 26 or more, most particularly 41 or more.Further, the upper limit thereof is not particularly limited, but ispreferably 652 or less. The expression “specifically hybridize with anmRNA consisting of the base sequence of SEQ ID NO: 5 in the sequencelisting” as used herein means that a polynucleotide does not hybridizewith mRNAs derived from a healthy person, but will hybridize with themRNA consisting of the base sequence of SEQ ID NO: 1 in the sequencelisting, under the conditions described in Example 1(4).

[0079] The plasmid of the present invention is not particularly limited,so long as it comprises the novel gene of the present invention, i.e.,the first novel gene of the present invention, the second novel gene ofthe present invention, or the third novel gene of the present invention.For example, it may be a respective plasmid prepared by incorporatingeach of the above genes of the present invention into a known vectorsuitably selected depending on a host cell used, that is, the firstplasmid of the present invention comprising the first novel gene of thepresent invention, the second plasmid of the present inventioncomprising the second novel gene of the present invention, and the thirdplasmid of the present invention comprising the third novel gene of thepresent invention.

[0080] The transformant of the present invention is not particularlylimited, so long as it comprises the plasmid of the present invention,i.e., the first plasmid of the present invention, the second plasmid ofthe present invention, or the third plasmid of the present invention.For example, it may be a transformant prepared by transforming a desiredhost cell with each of the plasmids of the present invention, that is,the first transformant comprising the first plasmid of the presentinvention, the second transformant comprising the second plasmid of thepresent invention, or the third transformant comprising the thirdplasmid of the present invention.

[0081] The host cell may be, for example, a known microorganism usuallyused, for example, E. coli or Saccharomyces cerevisiae, or a knowncultivated cell, such as an animal cell, such as a CHO cell or a COScell, or an insect cell such as a BmN4 cell.

[0082] The known expression vector may be, for example, pUC, pTV, PGEX,pKK, or pTrcHis for E. coli; pEMBLY or pYES2 for a yeast; pMAMneo for aCHO cell; pcDNA3 for a COS cell; a vector (such as pBK283) containing apolyhedrin promoter of a silkworm nucleopolyhederovirus (BmNPV).

[0083] The first antibody of the present invention or the fragmentthereof is reacted specifically with the first protein of the presentinvention or the variation functionally equivalent thereto,respectively. The second antibody of the present invention or thefragment thereof is reacted specifically with the second protein of thepresent invention or the variation functionally equivalent thereto,respectively. The third antibody of the present invention or thefragment thereof is reacted specifically with the third protein of thepresent invention or the variation functionally equivalent thereto,respectively.

[0084] The antibody of the present invention may be a monoclonalantibody or a polyclonal antibody.

[0085] The respective monoclonal antibodies of the present invention,that is, the first monoclonal antibody of the present inventionspecifically reactive with the first protein or the variationfunctionally equivalent thereto of the present invention, respectively,the second monoclonal antibody of the present invention specificallyreactive with the second protein or the variation functionallyequivalent thereto of the present invention, respectively, or the thirdmonoclonal antibody of the present invention specifically reactive withthe third protein or the variation functionally equivalent thereto ofthe present invention, respectively, may be prepared by a method whichis in itself known, except that the novel protein of the presentinvention, the variation functionally equivalent thereto, or a fragmentthereof is used as an immunogen or an antigen for a screening.

[0086] For example, a hybridoma secreting the monoclonal antibody of thepresent invention may be prepared by immunizing a mouse with the aboveimmunogen, fusing a splenic cell taken from the immunized mouse and amouse myeloma cell in accordance with a cell-fusion method disclosed inNature, 256, 495 (1975), or an electric cell-fusion method disclosed inJ. Immunol. Method, 100, 181-189 (1987), and carrying out a screeningwith the antigen for a screening as above.

[0087] As the medium for cultivating the hybridomas, any medium suitablefor a cultivation of a hybridoma may be used. Preferably, the Dulbecco'smodified Eeagle's minimum essential medium containing fetal calf serum,L-glutamine, L-pyruvic acid, and antibiotics (penicillin G andstreptomycin) may be used.

[0088] The cultivation of the hybridoma may be carried out in 5% CO₂ andat 37° C. for about 3 days in a medium, or for about 14 days in theabdominal cavities of mice.

[0089] It is possible to isolate or purify the monoclonal antibody fromthe resulting culture liquid or mouse ascites, using a method generallyapplied for the isolation and purification of proteins.

[0090] As examples thereof, there may be mentioned ammonium sulfatesalting out, ion exchange column chromatography using ion exchangecellulose, molecular sieve column chromatography using molecular sievegel, affinity column chromatography using protein A bindingpolysaccharides, dialysis, lyophilization, or the like.

[0091] The respective polyclonal antibodies of the present invention,that is, the first polyclonal antibody of the present inventionspecifically reactive with the first protein or the variationfunctionally equivalent thereto of the present invention, respectively,the second polyclonal antibody of the present invention specificallyreactive with the second protein or the variation functionallyequivalent thereto of the present invention, respectively, or the thirdpolyclonal antibody of the present invention specifically reactive withthe third protein or the variation functionally equivalent thereto ofthe present invention, respectively, may be also prepared by a methodwhich is in itself known, except that the novel protein of the presentinvention, the variation functionally equivalent thereto, or a fragmentthereof is used as an immunogen or an antigen for a screening by, forexample, the following method.

[0092] That is, a physiological salt solution containing an antigen ismixed with an equal volume of complete Freund's adjuvant or incompleteadjuvant, or an equivalent thereof, such as Hunter's TiterMax™(Funakoshi; Cat. No. YT001-00, Tokyo, Japan), until emulsified. Theresulting emulsion is administered subcutaneously, intraperitoneally, orintramuscularly to a mammal, for example, a rabbit or goat (a firstimmunization). Then, the same procedure is repeated at intervals of twoto four weeks for several immunizations. One or two weeks after a finalimmunization, blood is taken from a carotid artery or a heart of themammal, and salted-out with ammonium sulfate to prepare a serum.

[0093] Each of the antibody-fragments of the present invention is notparticularly limited, so long as it is a partial fragment of theantibody (including the monoclonal antibody and the polyclonal antibody)of the present invention, and has a specific reactivity that is the sameas that of the original antibody. The fragment of the present inventionmay be, for example, Fab, Fab′, F(ab′)₂, or Fv. The antibody fragment ofthe present invention may be prepared, for example, by digesting thepolyclonal antibody or monoclonal antibody of the present invention witha known protease by a conventional method, and then isolating andpurifying by a conventional method.

[0094] The inventors of the present invention found that the protein ofthe present invention, particularly, the protein consisting of the aminoacid sequence of SEQ ID NO: 2 in the sequence listing, the proteinconsisting of the amino acid sequence of SEQ ID NO: 4 in the sequencelisting, or the protein consisting of the amino acid sequence of SEQ IDNO: 6 in the sequence listing, and the mRNA thereof, is not expressed ina healthy person but in a patient suffering from a bacterial infection,such as septicemia, pneumonia, urinary tract infection, myelitis, ortympanitis. Therefore, the proteins of the present invention or mRNAsthereof may be used as a diagnostic marker of a patient suffering from abacterial infection. More particularly, when an in vitro detectionmethod of the present invention finds an existence of the protein of thepresent invention and/or mRNA thereof in a sample taken from a subject,the subject can be judged to be a patient suffering from a bacterialinfection. On the contrary, when the protein and/or the mRNA is notfound, the subject can be judged to be a person not suffering from abacterial infection.

[0095] The sample which may be used in the present invention is notparticularly limited, so long as it has a possibility of including theprotein of the present invention and/or the mRNA thereof. The sample maybe a biological sample taken from an animal, such as a mammal,particularly a human (particularly a patient), for example, a tissue(e.g., cells) or an extract therefrom, blood such as serum or plasma,urine, or a humor such as cerebrospinal fluid. A sample used in aconventional clinical examination may be used in the present inventionwithout limitation.

[0096] The method of the present invention will be explained hereinafterwith respect to the method for detecting a bacterial infection byanalyzing the mRNA of the protein of the present invention, and then,with respect to the method for detecting a bacterial infection byanalyzing the protein of the present invention.

[0097] In the methods of the present invention, the method for detectinga bacterial infection by analyzing the mRNA of the protein of thepresent invention is not particularly limited but, for example, may be amethod comprising steps of bring a sample into contact with apolynucleotide comprising a base sequence complementary to the basesequence of the mRNA of the protein of the present invention; andanalyzing a coupled product of the polynucleotide and the mRNA of theprotein of the present invention (hereinafter referred to as a “firstdetecting method of the present invention”), or a method comprising thesteps of reverse-transcribing an mRNA in a sample to a cDNA, amplifyinggenes in accordance with a gene-amplifying reaction, particularly, apolymerase chain reaction (PCR), using a reaction product obtained inthe reverse-transcribing step, and primers which may amplify genes withthe gene encoding the present protein as a template, and analyzing theamplified genes in the above gene-amplifying step (hereinafter referredto as a “second detecting method of the present invention”).

[0098] In the first method for detection of the present invention, thesample is reacted with a polynucleotide (for example, the probe of thepresent invention) comprising a base sequence complementary to that ofthe mRNA of the protein of the present invention, and the resultingcomplex of the polynucleotide and “the mRNA of the protein of thepresent invention” is detected, or the amount of the complex is measuredto thereby analyze the mRNA of the protein of the present invention.

[0099] The polynucleotide comprises a sequence complementary orsubstantially complementary to that of a part of the mRNA transcribedfrom a selected gene (DNA), and thus forms a double strand with the mRNAtranscribed from the target gene. It is believed that any polynucleotidesufficiently complementary to form a stable complex with a target mRNAcan be used. The polynucleotide able to be used in the present inventionmay be complementary to substantially any region in a target mRNA. Thepolynucleotide can be used as a DNA probe for detecting an increase or adecrease of an expression of the mRNA specific to the gene of theprotein according to the present invention. That is, the polynucleotideis specifically attached to the mRNA of the protein according to thepresent invention as a target, and forms a molecular hybrid, whereby adegree of expression of the mRNA of the protein according to the presentinvention in cells can be detected.

[0100] The polynucleotide able to be used in the first method fordetection of the present invention may be prepared by appropriatelyselecting a base sequence complementary to a specific base sequence ofthe mRNA of the protein according to the present invention, and using aknown DNA synthesizer, a PCR apparatus, a gene cloning or the like.Various length polynucleotides may be used, but the polynucleotidepreferably has 10 or more bases, more preferably 17 or more bases.

[0101] The polynucleotide may be a non-modified polynucleotide or apolynucleotide analogue. An appropriate analogue may be, for example, anethyl or methyl phosphate analogue, or a phosphorothioatedpolydeoxynucleotide [Nucleic Acids Res., 14, 9081-9093, (1986); J. Am.Chem. Soc., 106, 6077-6079, (1984)], with recent improvement in theproduction of polynucleotide analogue, for example, a2′-O-methylribonucleotide [Nucleic Acids Res., 15, 6131-6148, (1987)],or a conjugated RNA-DNA analogue, i.e., chimera polynucleotide [FEBSLett., 215, 327-330, (1987)], may be used.

[0102] The selected polynucleotide may be of any kind, for example, mayhave an electrical charge or no electrical charge. The polynucleotidemay be labeled with a known labeling agent, such as a radioactiveisotope, or a fluorescent substance by a conventional method, so as tocarry out the above experiment in vitro or in vivo. The radioactiveisotope may be, for example, ¹²⁵I, ¹³¹I, ³H, ¹⁴C, ³²P, or ³⁵S. Of theseradioactive isotopes, it is preferable to label the polynucleotide with³²P by a random primer method [Anal. Biochem., 132, 6-13, (1983)].Further, a fluorescent coloring agent forming a derivative may be usedas a labeling agent, as this enables an easy handling with a low riskfactor. As the fluorescent coloring agent, any coloring agents capableof binding the polynucleotide may be used. For example, fluorescein,rhodamin, Texas red, 4-fluoro-7-nitrobenzofurazane (NBD), coumarin,fluorescamine, succinyl fluorescein, or dansyl may be preferably used.

[0103] An amount of an mRNA of the protein according to the presentinvention may be measured by a northern blotting method, using cDNA ofthe protein according to the present invention as follows: an mRNA isextracted and isolated from any somatic cell or tissue, then theisolated mRNA is electrophoresed on an agarose gel and transferred ontoa nitro cellulose or nylon membrane, and then reacted with a cDNA probeof the protein according to the present invention to measure an amountof the mRNA of the protein according to the present invention. The cDNAprobe of the protein according to the present invention as used is a DNAcomplementary to the mRNA of the protein according to the presentinvention, and has preferably 17 or more bases.

[0104] In the reverse-transcribing step and the gene-amplifying step(particularly, the PCR step) of the second detecting method according tothe present invention, the reactions per se may be carried out inaccordance with the conventional reverse-transcribing method and theconventional gene-amplifying method, for example, areverse-transcription PCR (RT-PCR). More particularly, areverse-transcriptase and oligo(dT) primers are used to carry out thereverse-transcription. Then, a thermostable DNA polymerase, such as aTaq polymerase, is used to carry out an initial denaturing reaction, forexample at 97° C. for 2 to 3 minutes. Subsequently, an amplifying cycleconsisting of (1) a step for denaturing DNAs at 90 to 94° C. for 30seconds, (2) a step for annealing single-strand DNAs and primers at 50to 55° C. for 30 seconds, and (3) a step for synthesizing DNAs by thethermostable DNA polymerase at 70 to 75° C. for 1 to 2 minutes isrepeated, for example 15 to 45 times, to perform the PCR.

[0105] The analyzing step of the second detecting method according tothe present invention can be carried out in accordance with, forexample, a conventional analyzing method, for example, a methodcomprising steps of carrying out agarose-gel electrophoresis and thenstaining the gel with a suitable DNA-binding colorant such as ethidiumbromide, or a southern blotting, or the like

[0106] The method for detecting a bacterial infection by analyzing theprotein of the present invention, one of the methods according to thepresent invention, is not particularly limited but, for example, maycomprise steps of bringing a sample into contact with an immunoreactivesubstance which can immunologically react with the protein of thepresent invention, and analyzing a coupled product of the immunoreactivesubstance and the protein of the present invention (hereinafter referredto as a “third detecting method of the present invention”).

[0107] In the third detecting method of the present invention, thesample is brought into contact with the immunoreactive substance whichmay immunologically react with the protein of the present invention.When a sample from a human is used, the sample is preferably broughtinto contact with an immunoreactive substance which can immunologicallyreact with the protein consisting of the amino acid sequence of SEQ IDNO: 2, the protein consisting of the amino acid sequence of SEQ ID NO:4, or the protein consisting of the amino acid sequence of SEQ ID NO: 6.

[0108] When the sample is brought into contact with the substanceimmunologically reactive to the protein of the present invention, if thesample does not contain the protein of the present invention, a reactionwith the immunologically reactive substance does not occur. If thesample contains the protein of the present invention, theimmunologically reactive substance binds the protein of the presentinvention, and a complex of the immunologically reactive substance andthe protein of the present invention is formed in an amount correlatedwith that of the protein of the present invention present in the sample.The complex may be easily detected by a known method, and therefore, anexistence of the protein of the present invention in the sample can bedetected by detecting the existence of the complex, or an amount of theprotein of the present invention in the sample can be measured bymeasuring the amount of the complex. The protein of the presentinvention in a tissue or a cell may be measured by using a tissuesection sample or a cell sample in a fluorescent antibody technique oran enzyme antibody technique.

[0109] The immunologically reactive substance capable of immunologicallyreacting the protein of the present invention includes an antiserumagainst the protein of the present invention, a polyclonal antibodyagainst the protein of the present invention, or a monoclonal antibodyagainst the protein of the present invention, or a fragment of theseantibodies. The immunologically reactive substance may be used singly orin a combination thereof. The fragment includes, for example, Fab, Fab′,F(ab′)₂, or Fv.

[0110] In the third method for detection according to the presentinvention, the sample is brought into contact with the immunologicallyreactive substance capable of immunologically reacting the protein ofthe present invention, and a complex of the protein in the presentinvention and the immunologically reactive substance is formed. Then,the protein in the present invention bound to the antibody is detectedand the amount thereof is measured by an immunochemical method, tothereby find a level of the protein of the present invention in thesample.

[0111] Principally, the immunochemical method may be, for example, anyconventional immunoassay, for example, EIA, ELISA, RIA or the like. Theimmunochemical methods are generally classified as follows:

[0112] (1) Competitive Assay:

[0113] A sample containing an unknown amount of antigens and a givenamount of labeled antigens is competitively reacted with a given amountof antibodies, and then an activity of the labeled antigens bound to theantibodies or an activity of the labeled antigens not bound to theantibodies is measured.

[0114] (2) Sandwich Assay:

[0115] An excess amount of antibodies immobilized on carriers is addedand reacted to a sample containing an unknown amount of antigens (afirst reaction). Then, a given excess amount of labeled antibodies isadded and reacted therewith (a second reaction). An activity of thelabeled antibodies on the carriers is measured. Alternatively, anactivity of the labeled antibodies which are not on the carriers ismeasured. The first reaction and the second reaction may be carried outat the same time, or sequentially.

[0116] When a labeling agent is a radioactive isotope, a well counter ora scintillation counter may be used for measurement. When the labelingagent is an enzyme, an enzymatic activity can be measured by colorimetryor fluorimetry, after adding a substrate and allowing to stand. When thelabeling agent is a fluorescent substance or an luminescent substance, aknown method therefor may be used, respectively.

[0117] Recently, in addition to the above methods, a western blottingmethod has been used wherein electrophoresed proteins are transferredonto a filter such as a nitrocellulose membrane, and a target protein isdetected with an antibody. The western blotting method may also be usedin the detection of the protein according to the present invention.

[0118] The antibody used in the above methods can be labeled with anappropriate marker. Examples are a radioactive isotope, an enzyme, afluorescent substance, or a luminescent substance, by a known method oflabeling antibodies.

[0119] The radioactive isotope may be, for example, ¹²⁵I, ¹³¹I, ³H, ¹⁴C,or ³⁵S.

[0120] Preferably, the enzyme used is stable and has a large specificactivity. Examples of the enzyme are a glycosidase (such as,β-galactosidase, β-glucosidase, β-glucuronidase, β-fructosidase,α-galactosidase, α-glucosidase, or α-mannosidase), an amylase (such as,α-amylase, β-amylase, isoamylase, glucoamylase, or taka-amylase), acellulase, or a carbohydrase such as lysozyme; a urease, or an amidasesuch as asparaginase; a choline esterase, such as acetylcholinesterase,a phosphatase, such as alkaline phosphatase, a sulfatase, an esterasesuch as lipase; a nuclease such as deoxyribonuclease or ribonuclease; aniron porphyrin enzyme, such as a catalase, peroxidase or cytochromeoxidase; a copper enzyme, such as a tyrosinase or ascorbate oxidase;dehydrogenase, such as an alcohol dehydrogenase, malate dehydrogenase,lactate dehydrogenase, or isocitrate dehydrogenase.

[0121] The fluorescent substance may be, for example, fluorescamine, ora fluorescence isothiocyanate, and the luminescent substance may be, forexample, luminol, a luminol derivative, luciferin or lucigenin. A signalfrom the above label may be detected by known methods.

[0122] The labeling agent can be bound to antibodies by any conventionalmethod, such as a chloramin T method [Nature, 194, 495-496, (1962)], aperiodic acid method [Journal of Histochemistry and Cytochemistry, 22,1084-1091, (1974)], or a maleimide method [Journal of Biochemistry, 79,233-236, (1976)].

[0123] An EIA method, as one of the above measurement methods will bementioned hereinafter. A sample is added to the first antibodiesimmobilized on a carrier (such as an assay plate), and the firstantibodies are bound to the proteins of the present invention to formcomplexes. To the complexes, the second antibodies labeled with enzyme(such as peroxidase) are added to react with the complexes to form“first antibody/protein of the present invention/second antibody”complexes. To the resulting “first antibody/protein of the presentinvention/second antibody” complexes, a substrate for the enzyme label(such as peroxidase) is added, and an absorbance or fluorescent strengthof products of the enzymatic reaction is measured, whereby enzymaticactivities of the enzyme labels attached to the “first antibody/proteinin the present invention/second antibody” complexes are measured. Aseries of the above procedures is carried out in advance for a standardsolution containing a known amount of the protein of the presentinvention, and a standard curve based on the relationship between theprotein of the present invention and the absorbance or fluorescentstrength is prepared. A comparison is made between the standard curveand absorbance or fluorescent strength for a sample containing anunknown amount of the proteins according to the present invention, andthus, the amount of the proteins according to the present invention inthe sample can be measured.

[0124] Another EIA method will be mentioned hereinafter. A sample isbrought into contact with a carrier (such as an assay plate) toimmobilize the proteins of the present invention in the sample on thecarrier. Then, the first antibodies are added thereto to form complexesof the protein according to the present invention and the firstantibody. To the complexes are added anti-first antibody antibodies(second antibodies) labeled with an enzyme (such as peroxidase), toreact with the complexes to form “protein of the present invention/firstantibody/second antibody” complexes. To the resulting “protein of thepresent invention/first antibody/second antibody” complexes is added asubstrate for the enzyme label (such as peroxidase), and the absorbanceor fluorescent strength of products of the enzymatic reaction ismeasured, whereby enzymatic activities of the enzyme labels attached tothe “protein of the present invention/first antibody/second antibody”complexes are measured. A series of the above procedures is carried outin advance for a standard solution containing a known amount of theprotein according to the present invention, and a standard curve basedon the relationship between the protein of the present invention and theabsorbance or fluorescent strength is prepared. A comparison is madebetween the standard curve and the absorbance or fluorescent strengthfor a sample containing an unknown amount of the proteins according tothe present invention, and the amount of the proteins according to thepresent invention in the sample is measured.

[0125] Further, an RIA method will be mentioned hereinafter. A sample isadded to the first antibodies immobilized on a carrier (such as a testtube), and the first antibodies are bound to the proteins of the presentinvention to form complexes. To the complexes are added the secondantibodies labeled with radioactive isotope (such as ¹²⁵I), to reactwith the complexes to form “first antibody/protein of the presentinvention/second antibody” complexes. A radioactivity (such asγ-radioactivity) of the resulting “first antibody/protein of the presentinvention/second antibody” complexes is measured. A series of the aboveprocedures is carried out in advance for a standard solution containinga known amount of the protein according to the present invention, and astandard curve based on the relationship between the protein of thepresent invention and the radioactivity is prepared. A comparison ismade between the standard curve and the radioactivity for a samplecontaining an unknown amount of the proteins according to the presentinvention, and the amount of the proteins according to the presentinvention in the sample is measured.

[0126] Another RIA method will be mentioned hereinafter. A sample isbrought into contact with a carrier (such as a test tube) to immobilizethe proteins of the present invention in the sample on the carrier.Then, the first antibodies are added thereto to form complexes of theprotein according to the present invention and the first antibody. Tothe complexes are added anti-first antibody antibodies (secondantibodies) labeled with a radioactive isotope (such as ¹²⁵I), to reactwith the complexes to form “protein of the present invention/firstantibody/second antibody” complexes. A radioactivity (such asγ-radioactivity) of the resulting “protein of the presentinvention/first antibody/second antibody” complexes is measured. Aseries of the above procedures is carried out in advance for a standardsolution containing a known amount of the protein according to thepresent invention, and a standard curve based on the relationshipbetween the protein in the present invention and the radioactivity isprepared. A comparison is made between the standard curve and theradioactivity for a sample containing an unknown amount of the proteinsaccording to the present invention, and the amount of the proteinsaccording to the present invention in the sample is measured.

EXAMPLES

[0127] The present invention now will be further illustrated by, but isby no means limited to, the following Examples.

Example 1 Isolation and Identification of Activated HumanMacrophage-Specific Novel Genes

[0128] (1) Preparation of mRNA Derived from Macrophages Stimulated byLipopolysaccharide (LPS)

[0129] From 1 liter of blood derived from a healthy person as a startingmaterial, peripheral blood monocytes were prepared using a commerciallyavailable reagent for preparing peripheral blood monocytes (Lymphoprep;Nycomed, Oslo, Norway). The obtained peripheral blood monocytes weresuspended in an RPM1640 medium containing 10 μg/mL LPS (DifcoLaboratories, Detroit, Mich., USA) and 10% fetal calf serum (FCS) sothat the concentration of cells became 10⁶ cells/mL. To each plasticdish, 20 mL of the cell suspension was poured and cultured under thecondition of 37° C. and 5% CO₂.

[0130] After culturing for 3 hours, the supernatant was discarded, andadherent cells (i.e., macrophages stimulated by LPS) were washed threetimes with 20 mL of a phosphate-buffered saline (PBS). After 3 mL of asolution for cell lysis [4 mol/L guanidine isothiocyanate, and 30 mmol/Lsodium acetate (pH 4.8)] was added, suction and ejection were repeatedthree times using a syringe with a needle. The lysate was put on 1.2 mLof a 5.7 mol/L cesium chloride buffer (pH 4.8) in a ultracentrifuge 5PAtube (Hitachi Koki; Katsuta, Japan). After centrifuging for 18 hours(20° C., 38000 rpm), the supernatant in the centrifuge tube wasdiscarded. The pellet in the centrifuge tube was dissolved in 200 μL ofsterile water to collect RNA. From 1 L of blood, approximately 1 mg oftotal RNA (i.e., total RNA derived from macrophages stimulated by LPS)was obtained.

[0131] Then, using a commercially available kit for preparing mRNA[Poly(A) Quik mRNA Isolation Kit; Stratagene, La Jolla, Calif., USA], 15μg of mRNA (i.e., mRNA derived from macrophages stimulated by LPS) wasprepared from 500 μg of the total RNA.

[0132] (2) Preparation of a Phage cDNA Library

[0133] A phage cDNA library was prepared using 5 μg of the obtained mRNA(15 μg) derived from macrophages stimulated by LPS. Commerciallyavailable kits (ZAP Express cDNA Synthesis Kit and ZAP Express cDNAGigapack III Gold Cloning Kit; Stratagene) were used to prepare thephage cDNA library.

[0134] (3) Analysis of Partial Base Sequences of cDNAs

[0135] To analyze base sequences of cDNAs derived macrophages stimulatedby LPS, approximately 1000 phage plaques were picked up at random andcDNAs were recovered as a phagemid by a conventional method. Withrespect to the recovered approximately 1000 cDNAs derived macrophagesstimulated by LPS, 400 to 500 bases from the 5′ terminus and 3′ terminusof the cDNAs were analyzed, respectively, using a commercially availablekit for determining base sequences (Dye Terminator Cycle Sequencing kit;Perkin Elmer Japan, Urayasu). The DAN homology search of the obtainedsequences was carried out using BLAST (basic local alignment tool) inNCBI (National Center for Biotechnology Information;http://inhouse.ncbi.nlm.nih.gov) and found 63 unknown novel genes.

[0136] (4) Analysis by Northern Blotting

[0137] Total RNA derived macrophages stimulated by LPS were prepared inaccordance with the procedure for preparing total RNA derivedmacrophages stimulated by LPS described in Example (1). Further, totalRNAs derived macrophages not stimulated by LPS were prepared inaccordance with the procedure for preparing total RNA derivedmacrophages stimulated by LPS described in Example (1), except for usingan RPM1640 medium containing 10% FCS instead of the RPM1640 mediumcontaining 10 μg/mL LPS and 10% FCS. The total RNA derived macrophagesstimulated by LPS (10 μg/mL LPS) and the total RNA derived macrophagesnot stimulated by LPS were electrophoresed on a formaldehyde/agarose geland transferred onto a nylon membrane filter by a conventional method.

[0138] The filter onto which the RNAs were transferred was heat-treatedat 80° C. under reduced pressure for 2 hours, and immersed in acommercially available solution for prehybridization (Hybrisol I; Oncor,Gaithersburg, Md., USA) to perform prehybridization at 42° C. for 3hours. Then, the novel genes obtained in Example 1(3) labeled withisotope ³²P using a random primed labeling kit (Boehringer Mannheim;Germany) were respectively added, and hybridization was carried outovernight at 42° C. On the next day, the filter was twice washed with2×SSC (standard sodium citrate) containing 0.1% sodium dodecyl sulfate(SDS) at room temperature for 20 minutes, and further, twice washed with0.2×SSC containing 0.1% SDS at 65° C. for 20 minutes. The washed filterwas wrapped in a wrap, and autoradiography was performed overnight at−80° C.

[0139] As a result, it was found that, among 63 novel genes obtained inExample 1(3), three genes were those whose expression was induced by theLPS-stimulation. The results of northern blotting with respect to thethree novel genes (NLG-1-1, NLG-1-2, and NLG-2) are shown in FIG. 1. InFIG. 1, the symbol “+” means “stimulated by LPS”, the symbol “−” means“not stimulated by LPS”, and “Origin” means “starting point ofelectrophoresis”. The lengths of the mRNAs of the three novel genes(NLG-1-1, NLG-1-2, and NLG-2) were approximately 2.3 kb, approximately2.3 kb, and approximately 0.7 kb, respectively.

[0140] Further, the tissue-specific expression was examined. As shown inFIG. 2, the genes NLG-1-1 and NLG-1-2 were weakly expressed in alltissues examined [i.e., spleen (lane 1), thymus (lane 2), prostate (lane3), testis (lane 4), ovary (lane 5), small intestine (lane 6), largeintestine (lane 7) and peripheral blood lymphocyte (lane 8)]. On thecontrary, the gene NLG-2 was strongly expressed in testis (lane 4) andlarge intestine (lane 7), but not expressed in the other tissues.

[0141] (5) Determination of Full-Length Base Sequences

[0142] The full-length base sequences of the three novel genes (LG-1-1,NLG-1-2, and NLG-2) were determined by a conventional method.

[0143] The genes NLG-1-1 and NLG-1-2 consist of 2180 bp and 1970 bp,respectively. The concrete base sequences thereof are those of SEQ IDNO: 1 and SEQ ID NO: 3 in the sequence listing, respectively. As theresult of a homology search of the genes NLG-1-1 and NLG-1-2, the 193rdto 2139th base sequence of the gene NLG-1-1 was found to be completelyidentical to the 9th to 1955th base sequence of the gene NLG-1-2. It ispresumed that two mRNAs are transcribed from a chromosomal gene inaccordance with an alternative splicing. The gene NLG-1-1 encodes aprotein consisting of 481 amino acid residues having the amino acidsequence of SEQ ID NO: 2 in the sequence listing. The gene NLG-1-2encodes a protein consisting of 390 amino acid residues having the aminoacid sequence of SEQ ID NO: 4 in the sequence listing.

[0144] Further, the gene NLG-2 consists of 652 bp, and the concrete basesequence thereof is that of SEQ ID NO: 5 in the sequence listing. Thegene NLG-2 encodes a protein consisting of 83 amino acid residues havingthe amino acid sequence of SEQ ID NO: 6 in the sequence listing.

Example 2 Expression of the Genes NLG-1-1 and NLG-2 in an Animal Cell

[0145] In this example, the genes NLG-1-1 and NLG-2 were expressed usingCOS-1 (Dainippon pharmaceutical, Suita, Osaka, Japan) as an animal celland a pQBI25-fN3rsGFP vector (Quantum biotechnologies, Montreal, Quebec,Canada) by the following procedure. This was because when the abovevector is used, a desired gene can be expressed in the form of a fusedprotein with a green fluorescent protein (GFP), and therefore, locationsof the desired gene products can be observed by tracing the greenfluorescence.

[0146] Each cDNA of genes NLG-1-1 and NLG-2 was prepared by a reversetranscription PCR (RT-PCR) method in accordance with the followingprocedure. An mRNA was prepared from human peripheral blood monocytes(PBMC) stimulated by LPS for 3 hours. A cDNA synthesized from the mRNAusing a commercially available cDNA synthesis kit (SMART PCR cDNAsynthesis kit; Clontech, Palo alto, Calif., USA) was used as a template.

[0147] As primers, an NLG-2 forward primer consisting of the sequence:

[0148] 5′-CACGGATCCATTCTTCGCTGAAGTCATCATGAGC-3′ (SEQ ID NO: 7), an NLG-2reverse primer consisting of the sequence:

[0149] 5′-GTGGAATTCTTTGGTCACCCTTTGGACATTTTGC-3′ (SEQ ID NO: 8), anNLG-1-1 forward primer consisting of the sequence:

[0150] 5′-CACGGATCCTTCTTTACAACGAAATGATGCTCAAG-3′ (SEQ ID NO: 9), and anNLG-1-1 reverse primer consisting of the sequence:

[0151] 5′-GTGGAATTCGGAGAGATTTGTGATAGAATTATTACATGC-3′ (SEQ ID NO: 10)were used.

[0152] Using a commercially available reagent for PCR (Advantage cDNApolymerase Mix; Clontech), PCR was carried out by repeating a cycleconsisting of a denaturation step (94° C., 30 seconds) and an annealingand elongation step (68° C., 2 minutes), 30 times.

[0153] The obtained PCR product was digested with restriction enzymesBamHI (Takarashuzo, Chuo-ku, Tokyo, Japan) and EcoRI (Takarashuzo), andcloned in a pQBI25-fN3rsGFP vector using a commercially available kit(DNA ligation kit Ver. 2; Takarashuzo) to use for the followingexperiments.

[0154] On the day before a gene transfer, COS-1 cells were plated on a6-well plate so that the concentration of cells became 1×10⁶ cells/well.In each well of the 6-well plate, an autoclaved cover glass was placed,and cells were cultured on the cover glass. On the next day, thepreviously obtained vector was transferred to cos-1 cells using acommercially available reagent for transfection (LipofectAMINE reagent;Gibco BRL, Rockville, Md., USA). After 3 days from the transfer, cellson the cover glass were fixed in PBS containing 4% (v/v) formalin for 30minutes, treated in PBS containing 0.2% (v/v) Triton X-100 for 30minutes, and treated in a blocking reagent (Block Ace; Dainipponpharmaceutical) containing 20% (v/v) normal goat serum (VectorLaboratories, Burlingame, Calif., USA) for 30 minutes.

[0155] For immunostaining a mitochondrion, an anti-cytochrome c antibody(Santa Cruz Biotechnology, Santa Cruz, Calif., USA) and a Texasred-labeled anti-rabbit IgG antibody (Vector Laboratories) were used.For immunostaining an endoplasmic reticulum, an anti-calreticulinantibody (Upstate Biotechnology, Lake Placid, N.Y., USA) and a Texasred-labeled anti-rabbit IgG antibody (Vector Laboratories) were used.For immunostaining a Golgi apparatus, an anti-Golgi 58K protein antibody(Sigma, St. Louis, Mo., USA) and a Texas red-labeled anti-mouse IgGantibody (Kirkegaard & Perry Laboratories, Gaithersburg, Md., USA) wereused.

[0156] For staining a nucleus, propidium iodide (Wako Pure ChemicalIndustries, Osaka, Osaka, Japan) was used. For staining cytoplasm,hydroethidine (Polysciences, Warrington, Pa., USA) was used.

[0157] The cover glass was mounted on a slide glass, and observed with aconfocal laser scanning microscope FV500 (Olympus Optical CompanyLimited, Chiyoda-ku, Tokyo, Japan). The state of COS-1 cells in whichthe gene NLG-1-1 was expressed is shown in FIG. 3. The state of COS-1cells in which the gene NLG-2 was expressed is shown in FIG. 4. In FIGS.3 and 4, “A” is a green fluorograph showing the expression of the fusionprotein of GFP with the protein encoded by the gene NLG-1-1 or NLG-2;“B” is a red fluorograph wherein mitochondria were stained; “C” is acombination of the above fluorographs A and B; and “D” is a figure ofdifferential calculus interference.

[0158] As shown in FIG. 3A, the expression of the gene NLG-1-1 wasobserved around the nucleus. Further, the green fluorograph in FIG. 3Aand the red fluorograph (the fluorograph wherein mitochondria werestained) in FIG. 3B accorded well. In FIG. 3C, the areas whereinfluorographs A and B accorded were shown in yellow. On the contrary, thefluorograph wherein endoplasmic reticula were stained, the fluorographwherein Golgi apparatus were stained, the image wherein nuclei werestained, or the image wherein cytoplasm was stained did not accord withthe green fluorograph in FIG. 3A. Accordingly, it was found that theprotein encoded by the gene NLG-1-1 was localized in mitochondria.

[0159] Further, as apparent from FIG. 4, it was found that the proteinencoded by the gene NLG-2 was localized in mitochondria, as the proteinencoded by the gene NLG-1-1.

Example 3 Determination of the Chromosomal Locus of the Gene NLG-1-1

[0160] The chromosomal locus of the gene NLG-1-1 was determined by aFISH (fluorescence in situ hybridization) analysis [Chromosoma., 102,325-332 (1993)]. The gene was located on 13q22. The results were shownin FIG. 5. In FIG. 5, “A” is the result of DAPI(4′,6-diamidino-2-phenylindole) staining, and “B” is the result of theFISH signal. The arrow in FIG. 5A denotes the chromosomal locus of thegene NLG-1-1, and the number “13” denotes that it is the thirteenthchromosome.

Example 4 Determination of Expression of the Genes NLG-1-1, NLG-1-2, andNLG-2 in Patients Suffering from Septicemia

[0161] From 20 mL of each blood collected from four healthy persons andfour patients suffering from septicemia as a starting material,peripheral blood monocytes were prepared, and then approximately 20 μgof total RNA was respectively prepared from the peripheral bloodmonocytes in accordance with the procedure described in Example 1(1).

[0162] Using 10 μg of each RNA, northern blotting was carried out inaccordance with the procedure described in Example 1(4).

[0163] The results are shown in FIG. 6. In FIG. 6, (A) shows the resultusing the gene NLG-1-1 as a probe. Similarly, (B) and (C) show theresults using the genes NLG-1-2 and NLG-2 as a probe, respectively.Further, in FIG. 6, lanes 1 to 4 denote the results of RNAs derived fromthe healthy persons, and lanes 5 to 8 denote the results of RNAs derivedfrom the patients suffering from septicemia.

[0164] As shown in FIG. 6, three novel genes (NLG-1-1, NLG-1-2, andNLG-2) of the present invention were not expressed in all four healthypersons, but were strongly expressed in all four patients suffering fromsepticemia. These results show that these genes of the present inventionmay be used for the diagnosis of a bacterial infection or a judgment ofprognosis by analyzing the expression of the genes.

Industrial Applicability

[0165] It is known that life phenomena caused by an LPS-stimulation aresimilar to those in inflammation. In fact, the genes having anexpression that is induced by an LPS-stimulation specifically at amacrophage playing a central role in inflammation include almost allgenes encoding proteins leading to inflammatory diseases, for example,an inflammatory cytokine (TNF, IL-1, IL-6, IL-18), a chemokine (IL-8,MCP), a secretory protein, such as a collagenase abnormally produced ata diseased part of rheumatoid arthritis which is an inflammatorydisease, an NO synthase which is an intracellular protein and producesNO (nitrogen monoxide) causing an inflammation, cylooxygenese (COXII)producing a prostaglandin, an NF-kB, i.e., a gene-transcription factorparticipating in an expression of a gene encoding an inflammatoryprotein, or the like.

[0166] Clinical development for inhibitors of the above proteins as anantiinflammatory drug has been intensively carried out, and manyinhibitors are undergoing a clinical study. For example, an anti-TNFantibody or an inhibitor of NO synthase is already being used as amedicament for treating an inflammatory disease. Further, a clinicaldevelopment of a collagenase inhibitor as an inhibitor for a cancermetastasis, and of a COXII inhibitor as an anticancer drug is alreadybeing carried out. These suggest that there is a strong possibility thatthe novel genes having an expression that is induced by anLPS-stimulation and proteins according to the present invention alsoparticipate in the outbreak and/or deterioration of an inflammatorydisease, an allergy disease, or a cancer.

[0167] The possibility exists that the first and second novel proteinsof the present invention take part in an intracellular signaltransduction system of an LPS. Further, the possibility exists that thethird novel protein of the present invention takes part in anintracellular electron transportation and/or a radical production.Therefore, the proteins as above are different from a target of aconventional development of an antiinflammatory drug, whereas inhibitorsof the proteins as above will be a new type of antiinflammatory drug.Further, an examination of an expression of the genes encoding theproteins as above in a human clinical sample by means of a reversetranscription PCR (RT-PCR), a northern blotting, a dot blotting, or aDNA microarray would make it possible to carry out a diagnosis of aninflammation, an allergy, or a cancer. Furthermore, an antibody againstthe protein as above could be used to carry out a diagnosis of aninflammation, an allergy, or a cancer. Still further, antisense DNAs ofthe genes encoding the proteins as above could be used in a treatment ofan inflammation, an allergy, or a cancer (including a gene therapy).

[0168] The probe or the antibody according to the present invention maybe used to carry out a diagnosis of a bacterial infection, such assepticemia, pneumonia, urinary tract infection, myelitis, or tympanitis,or a judgment of prognosis. The proteins of the present invention areuseful for preparing the antibodies of the present invention, and thenovel genes, plasmids, and transformants of the present invention areuseful for preparing the proteins of the present invention.

[0169] As above, the present invention is explained with reference toparticular embodiments, but modifications and improvements obvious tothose skilled in the art are included in the scope of the presentinvention.

1 10 1 2180 DNA Homo sapiens CDS (37)..(1482) 1 ggcacgagct gaactgaacctcttctttac aacgaa atg atg ctc aag tct atc 54 Met Met Leu Lys Ser Ile 1 5aca gaa agc ttt gcc aca gca atc cat ggc ttg aaa gtg gga cac ctg 102 ThrGlu Ser Phe Ala Thr Ala Ile His Gly Leu Lys Val Gly His Leu 10 15 20 acagat cgt gtt att cag agg agc aag agg atg att cta gac act ctg 150 Thr AspArg Val Ile Gln Arg Ser Lys Arg Met Ile Leu Asp Thr Leu 25 30 35 ggt gctggg ttc ctg gga acc act acg gaa gtg ttt cac ata gcc agc 198 Gly Ala GlyPhe Leu Gly Thr Thr Thr Glu Val Phe His Ile Ala Ser 40 45 50 caa tat agcaag atc tac agt tcc aac ata tcc agc act gta tgg ggt 246 Gln Tyr Ser LysIle Tyr Ser Ser Asn Ile Ser Ser Thr Val Trp Gly 55 60 65 70 cag cca gacatc agg ctc ccg ccc aca tat gct gct ttt gtg aac ggt 294 Gln Pro Asp IleArg Leu Pro Pro Thr Tyr Ala Ala Phe Val Asn Gly 75 80 85 gtg gct att cactcc atg gat ttt gat gac acg tgg cac cct gcc acc 342 Val Ala Ile His SerMet Asp Phe Asp Asp Thr Trp His Pro Ala Thr 90 95 100 cac cct tct ggggct gtc ctt cct gtc ctc aca gct tta gca gaa gcc 390 His Pro Ser Gly AlaVal Leu Pro Val Leu Thr Ala Leu Ala Glu Ala 105 110 115 ctg cca agg agtcca aag ttt tct ggc ctt gac ctg ctg ctg gct ttc 438 Leu Pro Arg Ser ProLys Phe Ser Gly Leu Asp Leu Leu Leu Ala Phe 120 125 130 aat gtt ggt attgaa gtg caa ggc cga tta ctg cat ttc gcc aag gag 486 Asn Val Gly Ile GluVal Gln Gly Arg Leu Leu His Phe Ala Lys Glu 135 140 145 150 gcc aat gacatg cca aag aga ttc cat ccc cct tcc gtg gta gga acg 534 Ala Asn Asp MetPro Lys Arg Phe His Pro Pro Ser Val Val Gly Thr 155 160 165 ttg ggt agtgct gct gct gca tcc aag ttt tta gga ctt agc tcg aca 582 Leu Gly Ser AlaAla Ala Ala Ser Lys Phe Leu Gly Leu Ser Ser Thr 170 175 180 aag tgc cgagaa gct ctg gcc att gct gtt tcc cat gct ggg gca ccc 630 Lys Cys Arg GluAla Leu Ala Ile Ala Val Ser His Ala Gly Ala Pro 185 190 195 atg gcc aatgct gcc acc cag acc aag ccc ctc cac att ggc aat gct 678 Met Ala Asn AlaAla Thr Gln Thr Lys Pro Leu His Ile Gly Asn Ala 200 205 210 gcc aag catggg ata gaa gct gca ttt ttg gca atg ttg ggt ctc caa 726 Ala Lys His GlyIle Glu Ala Ala Phe Leu Ala Met Leu Gly Leu Gln 215 220 225 230 gga aacaag cag gtc ttg gac ttg gag gca gga ttt ggg gcc ttt tat 774 Gly Asn LysGln Val Leu Asp Leu Glu Ala Gly Phe Gly Ala Phe Tyr 235 240 245 gcc aactat tcc cca aaa gtc ctt cca agc ata gct tcc tac agt tgg 822 Ala Asn TyrSer Pro Lys Val Leu Pro Ser Ile Ala Ser Tyr Ser Trp 250 255 260 ctg ctggac cag cag gac gtg gcc ttt aag cgt ttt cct gca cat tta 870 Leu Leu AspGln Gln Asp Val Ala Phe Lys Arg Phe Pro Ala His Leu 265 270 275 tct acccac tgg gtg gca gac gca gct gca tct gtg aga aag cac ctt 918 Ser Thr HisTrp Val Ala Asp Ala Ala Ala Ser Val Arg Lys His Leu 280 285 290 gta gcagag aga gcc ctg ctt cca act gac tac att aag aga att gtg 966 Val Ala GluArg Ala Leu Leu Pro Thr Asp Tyr Ile Lys Arg Ile Val 295 300 305 310 ctcagg ata cca aat gtc cag tat gta aac agg ccc ttt cca gtt tcg 1014 Leu ArgIle Pro Asn Val Gln Tyr Val Asn Arg Pro Phe Pro Val Ser 315 320 325 gagcat gaa gcc cgt cat tca ttc cag tat gtg gcc tgt gcc atg ctg 1062 Glu HisGlu Ala Arg His Ser Phe Gln Tyr Val Ala Cys Ala Met Leu 330 335 340 cttgat ggt ggc atc act gtc ccc tca ttc cat gaa tgc cag atc aac 1110 Leu AspGly Gly Ile Thr Val Pro Ser Phe His Glu Cys Gln Ile Asn 345 350 355 aggcca cag gtg aga gag ctg ctc agt aag gtg gag ctg gag tac cct 1158 Arg ProGln Val Arg Glu Leu Leu Ser Lys Val Glu Leu Glu Tyr Pro 360 365 370 ccggac aac ttg cca agc ttc aac ata ctg tac tgt gaa ata agt gtc 1206 Pro AspAsn Leu Pro Ser Phe Asn Ile Leu Tyr Cys Glu Ile Ser Val 375 380 385 390acc ctc aag gat gga gcc acc ttc aca gat cgc tct gat acc ttc tat 1254 ThrLeu Lys Asp Gly Ala Thr Phe Thr Asp Arg Ser Asp Thr Phe Tyr 395 400 405ggg cac tgg aga aaa cca ctg agc cag gag gac cta gag gaa aag ttc 1302 GlyHis Trp Arg Lys Pro Leu Ser Gln Glu Asp Leu Glu Glu Lys Phe 410 415 420aga gcc aat gcc tcc aag atg ctg tcc tgg gac aca gtg gaa agc ctt 1350 ArgAla Asn Ala Ser Lys Met Leu Ser Trp Asp Thr Val Glu Ser Leu 425 430 435ata aag ata gtc aaa aat cta gaa gac cta gaa gac tgt tct gtg tta 1398 IleLys Ile Val Lys Asn Leu Glu Asp Leu Glu Asp Cys Ser Val Leu 440 445 450act aca ctt ctc aaa gga ccc tct cca cca gag gta gct tca aac tct 1446 ThrThr Leu Leu Lys Gly Pro Ser Pro Pro Glu Val Ala Ser Asn Ser 455 460 465470 cca gca tgt aat aat tct atc aca aat ctc tcc tgaggcttac caacatctaa1499 Pro Ala Cys Asn Asn Ser Ile Thr Asn Leu Ser 475 480 atgactttgcatttggggag attcaatgat ttggtttgta aagcaagggt ctgctgcttg 1559 gttttcccaggaaaaatgaa caaagatgga gagagtccag aaacagaact acatatatct 1619 ggaaggagccttctcctgaa aattttgcag gacagttcca cttacctaaa tcaagatgaa 1679 acacacacacaaaaatgagt ttgtaagcat tcacaagggt gaaattcaac tcacctgtga 1739 tttacttataaaattaatct cttcatagga attatgtgtg gacttcatga gcctcaaggt 1799 tttagagggatgtgaacctg catgtatatt ttctgacagt ggagagggct ctggtgcatt 1859 gtgtcaccaacagatctcct agaccatggc ttattaccaa gccctccaca gtgcaagggg 1919 tgctactggggaatgggtgg gtttaaatcc tgcctctgcc attcactaga tgtagccttg 1979 agcatgttaccattagccct ctgcctcagt ttccctattt gtcaagccga agtaaaaagc 2039 agtctggaaaaatcgcattt tggctctaga acccatggtc ttaagcactg caatatatca 2099 cctttcagtataaaaatatt tgaatcagag ttgcaataaa gaatgaaaag gaaaaaagag 2159 aagtaaaaaaaaaaaaaaaa a 2180 2 481 PRT Homo sapiens 2 Met Met Leu Lys Ser Ile ThrGlu Ser Phe Ala Thr Ala Ile His Gly 1 5 10 15 Leu Lys Val Gly His LeuThr Asp Arg Val Ile Gln Arg Ser Lys Arg 20 25 30 Met Ile Leu Asp Thr LeuGly Ala Gly Phe Leu Gly Thr Thr Thr Glu 35 40 45 Val Phe His Ile Ala SerGln Tyr Ser Lys Ile Tyr Ser Ser Asn Ile 50 55 60 Ser Ser Thr Val Trp GlyGln Pro Asp Ile Arg Leu Pro Pro Thr Tyr 65 70 75 80 Ala Ala Phe Val AsnGly Val Ala Ile His Ser Met Asp Phe Asp Asp 85 90 95 Thr Trp His Pro AlaThr His Pro Ser Gly Ala Val Leu Pro Val Leu 100 105 110 Thr Ala Leu AlaGlu Ala Leu Pro Arg Ser Pro Lys Phe Ser Gly Leu 115 120 125 Asp Leu LeuLeu Ala Phe Asn Val Gly Ile Glu Val Gln Gly Arg Leu 130 135 140 Leu HisPhe Ala Lys Glu Ala Asn Asp Met Pro Lys Arg Phe His Pro 145 150 155 160Pro Ser Val Val Gly Thr Leu Gly Ser Ala Ala Ala Ala Ser Lys Phe 165 170175 Leu Gly Leu Ser Ser Thr Lys Cys Arg Glu Ala Leu Ala Ile Ala Val 180185 190 Ser His Ala Gly Ala Pro Met Ala Asn Ala Ala Thr Gln Thr Lys Pro195 200 205 Leu His Ile Gly Asn Ala Ala Lys His Gly Ile Glu Ala Ala PheLeu 210 215 220 Ala Met Leu Gly Leu Gln Gly Asn Lys Gln Val Leu Asp LeuGlu Ala 225 230 235 240 Gly Phe Gly Ala Phe Tyr Ala Asn Tyr Ser Pro LysVal Leu Pro Ser 245 250 255 Ile Ala Ser Tyr Ser Trp Leu Leu Asp Gln GlnAsp Val Ala Phe Lys 260 265 270 Arg Phe Pro Ala His Leu Ser Thr His TrpVal Ala Asp Ala Ala Ala 275 280 285 Ser Val Arg Lys His Leu Val Ala GluArg Ala Leu Leu Pro Thr Asp 290 295 300 Tyr Ile Lys Arg Ile Val Leu ArgIle Pro Asn Val Gln Tyr Val Asn 305 310 315 320 Arg Pro Phe Pro Val SerGlu His Glu Ala Arg His Ser Phe Gln Tyr 325 330 335 Val Ala Cys Ala MetLeu Leu Asp Gly Gly Ile Thr Val Pro Ser Phe 340 345 350 His Glu Cys GlnIle Asn Arg Pro Gln Val Arg Glu Leu Leu Ser Lys 355 360 365 Val Glu LeuGlu Tyr Pro Pro Asp Asn Leu Pro Ser Phe Asn Ile Leu 370 375 380 Tyr CysGlu Ile Ser Val Thr Leu Lys Asp Gly Ala Thr Phe Thr Asp 385 390 395 400Arg Ser Asp Thr Phe Tyr Gly His Trp Arg Lys Pro Leu Ser Gln Glu 405 410415 Asp Leu Glu Glu Lys Phe Arg Ala Asn Ala Ser Lys Met Leu Ser Trp 420425 430 Asp Thr Val Glu Ser Leu Ile Lys Ile Val Lys Asn Leu Glu Asp Leu435 440 445 Glu Asp Cys Ser Val Leu Thr Thr Leu Leu Lys Gly Pro Ser ProPro 450 455 460 Glu Val Ala Ser Asn Ser Pro Ala Cys Asn Asn Ser Ile ThrAsn Leu 465 470 475 480 Ser 3 1970 DNA Homo sapiens CDS (126)..(1298) 3ggcacgaggc cagccaatat agcaagatct acagttccaa catatccagc actgtttggg 60gtcagccaga catcaggctc ccgcccacat atgctgcttt tgtgaacggt gtggctattc 120actcc atg gat ttt gat gac acg tgg cac cct gcc acc cac cct tct ggg 170Met Asp Phe Asp Asp Thr Trp His Pro Ala Thr His Pro Ser Gly 1 5 10 15gct gtc ctt cct gtc ctc aca gct tta gca gaa gcc ctg cca agg agt 218 AlaVal Leu Pro Val Leu Thr Ala Leu Ala Glu Ala Leu Pro Arg Ser 20 25 30 ccaaag ttt tct ggc ctt gac ctg ctg ctg gct ttc aat gtt ggt att 266 Pro LysPhe Ser Gly Leu Asp Leu Leu Leu Ala Phe Asn Val Gly Ile 35 40 45 gaa gtgcaa ggc cga tta ctg cat ttc gcc aag gag gcc aat gac atg 314 Glu Val GlnGly Arg Leu Leu His Phe Ala Lys Glu Ala Asn Asp Met 50 55 60 cca aag agattc cat ccc cct tcc gtg gta gga acg ttg ggt agt gct 362 Pro Lys Arg PheHis Pro Pro Ser Val Val Gly Thr Leu Gly Ser Ala 65 70 75 gct gct gca tccaag ttt tta gga ctt agc tcg aca aag tgc cga gaa 410 Ala Ala Ala Ser LysPhe Leu Gly Leu Ser Ser Thr Lys Cys Arg Glu 80 85 90 95 gct ctg gcc attgct gtt tcc cat gct ggg gca ccc atg gcc aat gct 458 Ala Leu Ala Ile AlaVal Ser His Ala Gly Ala Pro Met Ala Asn Ala 100 105 110 gcc acc cag accaag ccc ctc cac att ggc aat gct gcc aag cat ggg 506 Ala Thr Gln Thr LysPro Leu His Ile Gly Asn Ala Ala Lys His Gly 115 120 125 ata gaa gct gcattt ttg gca atg ttg ggt ctc caa gga aac aag cag 554 Ile Glu Ala Ala PheLeu Ala Met Leu Gly Leu Gln Gly Asn Lys Gln 130 135 140 gtc ttg gac ttggag gca gga ttt ggg gcc ttt tat gcc aac tat tcc 602 Val Leu Asp Leu GluAla Gly Phe Gly Ala Phe Tyr Ala Asn Tyr Ser 145 150 155 cca aaa gtc cttcca agc ata gct tcc tac agt tgg ctg ctg gac cag 650 Pro Lys Val Leu ProSer Ile Ala Ser Tyr Ser Trp Leu Leu Asp Gln 160 165 170 175 cag gac gtggcc ttt aag cgt ttt cct gca cat tta tct acc cac tgg 698 Gln Asp Val AlaPhe Lys Arg Phe Pro Ala His Leu Ser Thr His Trp 180 185 190 gtg gca gacgca gct gca tct gtg aga aag cac ctt gta gca gag aga 746 Val Ala Asp AlaAla Ala Ser Val Arg Lys His Leu Val Ala Glu Arg 195 200 205 gcc ctg cttcca act gac tac att aag aga att gtg ctc agg ata cca 794 Ala Leu Leu ProThr Asp Tyr Ile Lys Arg Ile Val Leu Arg Ile Pro 210 215 220 aat gtc cagtat gta aac agg ccc ttt cca gtt tcg gag cat gaa gcc 842 Asn Val Gln TyrVal Asn Arg Pro Phe Pro Val Ser Glu His Glu Ala 225 230 235 cgt cat tcattc cag tat gtg gcc tgt gcc atg ctg ctt gat ggt ggc 890 Arg His Ser PheGln Tyr Val Ala Cys Ala Met Leu Leu Asp Gly Gly 240 245 250 255 atc actgtc ccc tca ttc cat gaa tgc cag atc aac agg cca cag gtg 938 Ile Thr ValPro Ser Phe His Glu Cys Gln Ile Asn Arg Pro Gln Val 260 265 270 aga gagctg ctc agt aag gtg gag ctg gag tac cct ccg gac aac ttg 986 Arg Glu LeuLeu Ser Lys Val Glu Leu Glu Tyr Pro Pro Asp Asn Leu 275 280 285 cca agcttc aac ata ctg tac tgt gaa ata agt gtc acc ctc aag gat 1034 Pro Ser PheAsn Ile Leu Tyr Cys Glu Ile Ser Val Thr Leu Lys Asp 290 295 300 gga gccacc ttc aca gat cgc tct gat acc ttc tat ggg cac tgg aga 1082 Gly Ala ThrPhe Thr Asp Arg Ser Asp Thr Phe Tyr Gly His Trp Arg 305 310 315 aaa ccactg agc cag gag gac cta gag gaa aag ttc aga gcc aat gcc 1130 Lys Pro LeuSer Gln Glu Asp Leu Glu Glu Lys Phe Arg Ala Asn Ala 320 325 330 335 tccaag atg ctg tcc tgg gac aca gtg gaa agc ctt ata aag ata gtc 1178 Ser LysMet Leu Ser Trp Asp Thr Val Glu Ser Leu Ile Lys Ile Val 340 345 350 aaaaat cta gaa gac cta gaa gac tgt tct gtg tta act aca ctt ctc 1226 Lys AsnLeu Glu Asp Leu Glu Asp Cys Ser Val Leu Thr Thr Leu Leu 355 360 365 aaagga ccc tct cca cca gag gta gct tca aac tct cca gca tgt aat 1274 Lys GlyPro Ser Pro Pro Glu Val Ala Ser Asn Ser Pro Ala Cys Asn 370 375 380 aattct atc aca aat ctc tcc tgaggcttac caacatctaa atgactttgc 1325 Asn SerIle Thr Asn Leu Ser 385 390 atttggggag attcaatgat ttggtttgta aagcaagggtctgctgcttg gttttcccag 1385 gaaaaatgaa caaagatgga gagagtccag aaacagaactacatatatct ggaaggagcc 1445 ttctcctgaa aattttgcag gacagttcca cttacctaaatcaagatgaa acacacacac 1505 aaaaatgagt ttgtaagcat tcacaagggt gaaattcaactcacctgtga tttacttata 1565 aaattaatct cttcatagga attatgtgtg gacttcatgagcctcaaggt tttagaggga 1625 tgtgaacctg catgtatatt ttctgacagt ggagagggctctggtgcatt gtgtcaccaa 1685 cagatctcct agaccatggc ttattaccaa gccctccacagtgcaagggg tgctactggg 1745 gaatgggtgg gtttaaatcc tgcctctgcc attcactagatgtagccttg agcatgttac 1805 cattagccct ctgcctcagt ttccctattt gtcaagccgaagtaaaaagc agtctggaaa 1865 aatcgcattt tggctctaga acccatggtc ttaagcactgcaatatatca cctttcagta 1925 taaaaatatt tgaatcagag ttgcaataaa aaaaaaaaaaaaaaa 1970 4 390 PRT Homo sapiens 4 Met Asp Phe Asp Asp Thr Trp His ProAla Thr His Pro Ser Gly Ala 1 5 10 15 Val Leu Pro Val Leu Thr Ala LeuAla Glu Ala Leu Pro Arg Ser Pro 20 25 30 Lys Phe Ser Gly Leu Asp Leu LeuLeu Ala Phe Asn Val Gly Ile Glu 35 40 45 Val Gln Gly Arg Leu Leu His PheAla Lys Glu Ala Asn Asp Met Pro 50 55 60 Lys Arg Phe His Pro Pro Ser ValVal Gly Thr Leu Gly Ser Ala Ala 65 70 75 80 Ala Ala Ser Lys Phe Leu GlyLeu Ser Ser Thr Lys Cys Arg Glu Ala 85 90 95 Leu Ala Ile Ala Val Ser HisAla Gly Ala Pro Met Ala Asn Ala Ala 100 105 110 Thr Gln Thr Lys Pro LeuHis Ile Gly Asn Ala Ala Lys His Gly Ile 115 120 125 Glu Ala Ala Phe LeuAla Met Leu Gly Leu Gln Gly Asn Lys Gln Val 130 135 140 Leu Asp Leu GluAla Gly Phe Gly Ala Phe Tyr Ala Asn Tyr Ser Pro 145 150 155 160 Lys ValLeu Pro Ser Ile Ala Ser Tyr Ser Trp Leu Leu Asp Gln Gln 165 170 175 AspVal Ala Phe Lys Arg Phe Pro Ala His Leu Ser Thr His Trp Val 180 185 190Ala Asp Ala Ala Ala Ser Val Arg Lys His Leu Val Ala Glu Arg Ala 195 200205 Leu Leu Pro Thr Asp Tyr Ile Lys Arg Ile Val Leu Arg Ile Pro Asn 210215 220 Val Gln Tyr Val Asn Arg Pro Phe Pro Val Ser Glu His Glu Ala Arg225 230 235 240 His Ser Phe Gln Tyr Val Ala Cys Ala Met Leu Leu Asp GlyGly Ile 245 250 255 Thr Val Pro Ser Phe His Glu Cys Gln Ile Asn Arg ProGln Val Arg 260 265 270 Glu Leu Leu Ser Lys Val Glu Leu Glu Tyr Pro ProAsp Asn Leu Pro 275 280 285 Ser Phe Asn Ile Leu Tyr Cys Glu Ile Ser ValThr Leu Lys Asp Gly 290 295 300 Ala Thr Phe Thr Asp Arg Ser Asp Thr PheTyr Gly His Trp Arg Lys 305 310 315 320 Pro Leu Ser Gln Glu Asp Leu GluGlu Lys Phe Arg Ala Asn Ala Ser 325 330 335 Lys Met Leu Ser Trp Asp ThrVal Glu Ser Leu Ile Lys Ile Val Lys 340 345 350 Asn Leu Glu Asp Leu GluAsp Cys Ser Val Leu Thr Thr Leu Leu Lys 355 360 365 Gly Pro Ser Pro ProGlu Val Ala Ser Asn Ser Pro Ala Cys Asn Asn 370 375 380 Ser Ile Thr AsnLeu Ser 385 390 5 652 DNA Homo sapiens CDS (56)..(307) 5 ggcaccaggcgcaccgcccg gcgtccagat ttggcaattc ttcgctgaag tcatc atg 58 Met 1 agc tttttc caa ctc ctg atg aaa agg aag gaa ctc att ccc ttg gtg 106 Ser Phe PheGln Leu Leu Met Lys Arg Lys Glu Leu Ile Pro Leu Val 5 10 15 gtg ttc atgact gtg gcg gcg ggt gga gcc tca tct ttc gct gtg tat 154 Val Phe Met ThrVal Ala Ala Gly Gly Ala Ser Ser Phe Ala Val Tyr 20 25 30 tct ctt tgg aaaacc gat gtg atc ctt gat cga aaa aaa aat cca gaa 202 Ser Leu Trp Lys ThrAsp Val Ile Leu Asp Arg Lys Lys Asn Pro Glu 35 40 45 cct tgg gaa act gtggac cct act gta cct caa aag ctt ata aca atc 250 Pro Trp Glu Thr Val AspPro Thr Val Pro Gln Lys Leu Ile Thr Ile 50 55 60 65 aac caa caa tgg aaaccc att gaa gag ttg caa aat gtc caa agg gtg 298 Asn Gln Gln Trp Lys ProIle Glu Glu Leu Gln Asn Val Gln Arg Val 70 75 80 acc aaa tgacgagccctcgcctcttt cttctgaaga gtactctata aatctagtgg 354 Thr Lys aaacatttctgcacaaacta gattctggac accagtgtgc ggaaatgctt ctgctacatt 414 tttagggtttgtctacattt tttgggctct ggataaggaa ttaaaggagt gcagcaataa 474 ctgcactgtctaaaagtttg tgcttatttt cttgtaaatt tgaatattgc atattgaaat 534 ttttgtttatgatctatgaa tgtttttctt aaaatttaca aagctttgta aattagattt 594 tctttaataaaatgccattt gtgcaagatt tctcaaagaa aaaaaaaaaa aaaaaaaa 652 6 83 PRT Homosapiens 6 Met Ser Phe Phe Gln Leu Leu Met Lys Arg Lys Glu Leu Ile ProLeu 1 5 10 15 Val Val Phe Met Thr Val Ala Ala Gly Gly Ala Ser Ser PheAla Val 20 25 30 Tyr Ser Leu Trp Lys Thr Asp Val Ile Leu Asp Arg Lys LysAsn Pro 35 40 45 Glu Pro Trp Glu Thr Val Asp Pro Thr Val Pro Gln Lys LeuIle Thr 50 55 60 Ile Asn Gln Gln Trp Lys Pro Ile Glu Glu Leu Gln Asn ValGln Arg 65 70 75 80 Val Thr Lys 7 34 DNA Artificial Sequence Descriptionof Artificial Sequence NLG-2 forward primer 7 cacggatcca ttcttcgctgaagtcatcat gagc 34 8 34 DNA Artificial Sequence Description ofArtificial Sequence NLG-2 reverse primer 8 gtggaattct ttggtcaccctttggacatt ttgc 34 9 35 DNA Artificial Sequence Description ofArtificial Sequence NLG-1-1 forward primer 9 cacggatcct tctttacaacgaaatgatgc tcaag 35 10 39 DNA Artificial Sequence Description ofArtificial Sequence NLG-1-1 reverse primer 10 gtggaattcg gagagatttgtgatagaatt attacatgc 39

1. A protein comprising an amino acid sequence of SEQ ID NO: 2 in thesequence listing, or a variation functionally equivalent thereto, or afragment of said protein or said variation.
 2. A gene encoding saidprotein or said variation functionally equivalent thereto according toclaim
 1. 3. The gene according to claim 2, consisting of the 37th to1479th bases in a base sequence of SEQ ID NO: 1 in the sequence listing.4. A plasmid comprising said gene according to claim 2 or
 3. 5. Atransformant comprising said plasmid according to claim
 4. 6. Anantibody or a fragment thereof, characterized by being reactivespecifically to said protein or said variation functionally equivalentthereto according to claim
 1. 7. A method for detecting a bacterialinfection, characterized by analyzing a protein comprising an amino acidsequence of SEQ ID NO: 2 in the sequence listing or a variationfunctionally equivalent thereto, or an mRNA thereof in a sample to bedetected.
 8. The method according to claim 7, wherein the bacterialinfection is septicemia.
 9. A polynucleotide capable of specificallyhybridizing to an mRNA consisting of an base sequence of SEQ ID NO: 1 inthe sequence listing.
 10. A protein comprising an amino acid sequence ofSEQ ID NO: 4 in the sequence listing, or a variation functionallyequivalent thereto, or a fragment of said protein or said variation. 11.A gene encoding said protein or said variation functionally equivalentthereto according to claim
 10. 12. The gene according to claim 11,consisting of the 126th to 1295th bases in a base sequence of SEQ ID NO:3 in the sequence listing.
 13. A plasmid comprising said gene accordingto claim 11 or
 12. 14. A transformant comprising said plasmid accordingto claim
 13. 15. An antibody or a fragment thereof, characterized bybeing reactive specifically to said protein or said variationfunctionally equivalent thereto according to claim
 10. 16. A method fordetecting a bacterial infection, characterized by analyzing a proteincomprising an amino acid sequence of SEQ ID NO: 4 in the sequencelisting or a variation functionally equivalent thereto, or an mRNAthereof in a sample to be detected.
 17. The method according to claim16, wherein the bacterial infection is septicemia.
 18. A polynucleotidecapable of specifically hybridizing to an mRNA consisting of an basesequence of SEQ ID NO: 3 in the sequence listing.
 19. A proteincomprising an amino acid sequence of SEQ ID NO: 6 in the sequencelisting, or a variation functionally equivalent thereto, or a fragmentof said protein or said variation.
 20. A gene encoding said protein orsaid variation functionally equivalent thereto according to claim 19.21. The gene according to claim 20, consisting of the 56th to 304thbases in a base sequence of SEQ ID NO: 5 in the sequence listing.
 22. Aplasmid comprising said gene according to claim 20 or
 21. 23. Atransformant comprising said plasmid according to claim
 22. 24. Anantibody or a fragment thereof, characterized by being reactivespecifically to said protein or said variation functionally equivalentthereto according to claim
 19. 25. A method for detecting a bacterialinfection, characterized by analyzing a protein comprising an amino acidsequence of SEQ ID NO: 6 in the sequence listing or a variationfunctionally equivalent thereto, or an mRNA thereof in a sample to bedetected.
 26. The method according to claim 25, wherein the bacterialinfection is septicemia.
 27. A polynucleotide capable of specificallyhybridizing to an mRNA consisting of an base sequence of SEQ ID NO: 5 inthe sequence listing.